Template:Adding COVID-19 and other virus testing to your laboratory

3. Adding COVID-19 and other virus testing to your laboratory

Maybe you've been running an environmental health laboratory and want to expand into clinical health testing. Perhaps you're in charge of an academic research lab but want to expand to the clinical diagnostic side. Or maybe you're running a physician office laboratory (POL) and are wondering if it's even possible to expand your waived testing efforts to COVID-19. Where the previous chapter discussed the "what" of COVID-19 and viral testing, this chapter aims to help you with the "how" of adding it to your laboratory offerings.

Naturally, many questions come with the "how":

• Does using one method make the most sense, or will your lab turn to multiple methods for virus testing? This may be determined by current equipment, space considerations, and budget.
• What type of lab are you running? A POL is going to have fewer options available than a CLIA moderate- or high-complexity lab.
• How interoperable are you existing laboratory and clinical informatics solutions? Research laboratories face more challenges in integrating their systems with EHRs and other clinical systems.
• What vendors and consultants are out there to help get equipped? Some vendors have very specific solutions, whereas others may have a broader range of offerings.

These questions and more are addressed in this chapter.

3.1 What methodologies will you use?

3.1.1 PCR

SARS-CoV-2 PCR screening test by nasal swab in Strasbourg on August 21, 2020.

In the previous chapter, the most common testing methodologies for COVID-19 and other coronaviruses were discussed in detail. The prevailing method (often called the "gold standard") among them all is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for testing. Broadly speaking, PCR is useful in pharmaceutical, biotechnology, and genetic engineering endeavors, as well as clinical diagnostics. As such, labs in those industries that already have PCR infrastructure in place have a theoretical step-up over labs that do not.

PCR technology has advanced to the point where it is more efficient and user-friendly than prior, yet "the high cost of the instruments, servicing contracts, and reagents pose major challenges for the market, especially to the price-sensitive academics." Writing about the thirty-fifth anniversary of PCR in 2018, science writer Alan Dove not only highlighted these cost issues but also the size and energy requirements for running the equipment. "As a result, one of the defining techniques of modern molecular biology has remained stubbornly inaccessible to educators and unusable in many remote locations." Various efforts have been made over the years to bring costs down by modifying how heating and temperature control are performed, but many of those system aren't typically optimal during a pandemic when turnaround time is critical.

Amidst the pandemic, additional challenges also exist to those wanting to conduct PCR testing for COVID-19 and other viruses. As was discussed at the end of the previous chapter, supplies of reagents and consumables are not particularly robust mid-pandemic, with various shortages being reported off and on since the start of the pandemic. Some of these shortages have gradually worked themselves out over time, but they highlight the need for other varying methods that don't necessarily depend on the same reagents and consumables that are in short supply.

For those labs wishing to adopt PCR testing of viruses—particularly COVID-19—into their workflow while providing reasonable turnaround times, all is not lost. However, careful planning is required. For example, you'll want to keep in mind that some PCR machines require vendor-specific reagents. If you're going to acquire a particular instrument, you'll want to do due diligence by verifying not only the supported reagents but also those reagents' overall availability (real and projected). You'll also want to consider factors such as anticipated workload (tests per day), what your workflow will look like, and how to balance overall investment with the need for reasonable turnaround times.

An increasing body of research is being produced suggesting ways to improve turnaround times with PCR testing for COVID-19, with many research efforts focusing on cutting out RNA extraction steps entirely. Alcoba-Florez et al. propose direct heating of the sample-containing nasopharyngeal swab at 70 °C for 10 minutes in place of RNA extraction. Adams et al. have proposed an "adaptive PCR" method using a non-standard reagent mix that skips RNA extraction and can act "as a contingency for resource‐limited settings around the globe." Wee et al. skip RNA extraction and nucleic acid purification by using a single-tube homogeneous reaction method run on a lightweight, portable thermocycler. Other innovations include tweaking reagents and enzymes to work with one step, skipping the reverse transcription step, and using saliva-based molecular testing that skips RNA extraction.

Saliva as a specimen

The saliva molecular tests in particular are intriguing. Talk of the potential utility of using saliva as a specimen for COVID-19 was occurring as early as April 2020, and the first saliva-based COVID-19 test, produced by Spectrum Solutions in cooperation with RUCDR Infinite Biologics Laboratory and Vault Health, was given an FDA EUA in April 2020. On August 15, 2020, Yale School of Public Health was given an EUA for it SalivaDirect molecular test. Although still PCR-based (and a CLIA high-complexity test), SalivaDirect is being touted as a means to improve specimen collection safety, consume fewer reagents, prove compatible with high-throughput workflow, and cut overall turnaround time. Not only is saliva easier to collect and safer for healthcare staff, the test is essentially "open sourced," not requiring proprietary equipment from Yale, making the test more flexible by being validated to reliably function with a wider array of reagents and instruments. When compared to using a nasopharyngeal swab specimen using the ThermoFisher Scientific TaqPath COVID-19 combo kit, results were comparable 94.1% of the time. While sensitivity and specificity may be slightly less comparable to other PCR options, the overall advantages during reagent shortages and a definitive need for broader testing likely outweigh the slightly lesser sensitivity and specificity. In November 2020, public health agencies in Arizona and Minnesota reportedly began running trials of free saliva-based molecular testing.

As the pandemic has progressed into 2021, saliva testing has become even more attractive, in particular for at-home over-the-counter testing. In August 2021, Spectrum Solutions received an EUA for its Spectrum Solutions SDNA-1000 saliva collection system, specifically designed "to avoid user collection errors" and eliminate "the requirement for any bio-sample temperature-controlled storage or transport," arguably upping the game for new saliva-based test kits going forward. Additionally, as variants of COVID-19 continue to crop up, additional saliva-based at-home tests are coming into development. For example, researchers at the Wyss Institute, the Massachusetts Institute of Technology, and Boston-area hospitals have been working on a laboratory-developed test called Minimally Instrumented SHERLOCK (miSHERLOCK) based on CRISPR (clustered regularly interspaced short palindromic repeats) technology. The researchers claim that the test, able to be used with typical off-the-shelf components, "works as well as the gold standard PCR tests and could cost as little as $3 per test."

3.1.2 Pooled testing

Another method some labs are taking to speed up turnaround time is using pooled testing. The general concept involves placing two or more test specimens together and testing the pool as one specimen. The most obvious advantage to this is that the process saves on reagents and other supplies, particularly when supply chains are disrupted, and it reduces the amount of time required to analyze large quantities of specimens. This methodology is best used "in situations where disease prevalence is low, since each negative pool test eliminates the need to individually test those specimens and maximizes the number of individuals who can be tested over a given amount of time." However, it's best left to situations where expectations are that less than 10 percent of the population being tested is affected by what's being tested for.

The downside of pooled testing comes with the issues of dilution, contamination, and populations with 10 or more percent infected. A target-positive specimen that commingles with other target-free specimens is itself diluted and in some cases may cause issues with the limit of detection for the assay. Additionally, if the pool tests positive, target-free specimens may become contaminated by a target-positive specimen. This may cause issues with any individual specimen assays that get ran. And the workflows involving pooling must be precise, as a technician working with multiple specimens at the same time increases the chance of lab errors. Finally, at least in the U.S., a Food and Drug Administration (FDA) emergency use authorization (EUA) for a validated pooled testing method is required. (Validation of pooled methods may differ in other countries.) The U.S. Centers for Disease Control and Prevention (CDC) has published interim guidance on pooled testing strategies for SARS-CoV-2.

On April 20, 2021, the FDA updated its policies to allow for pooled testing to be added to the use case scenarios for several existing test kits. "This means that tests with EUAs that are amended by this authorization may be used with pooled anterior nasal specimens from individuals without known or suspected COVID-19 when such individuals are tested as part of a testing program that includes testing at regular intervals, at least once per week." However, affected kits can only be used in high-complexity CLIA labs, though "tests authorized for use in specific named or designated high-complexity laboratories can only be used in such laboratories." As of September 2021, four PCR test kit EUAs were amended to allow for pooled testing:

• Biomeme SARS-CoV-2 Real-Time RT-PCR Test
• Clinical Enterprise SARS-SoV-2-RT-PCR Assay
• CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay (Version 3)
• Viracor SARS-CoV-2 assay

3.1.3 Rapid antigen testing

As mentioned in the previous chapter, the benefits of antigen testing For COVID-19 and other viral infections are 1. specimen collection can typically be done with a simple nasal swab rather than a more invasive nasopharyngeal swab, 2. testing is more rapid and convenient, and 3. it takes some pressure off the PCR supply chain. However, antigen testing only tests what's there, rather than amplifying the amount, resulting in generally lower sensitivities. As such, the real utility of antigen testing, despite its lower sensitivity, appears to be surveillance situations where a large group of individuals who are at risk can be screened at regularly scheduled intervals of two to four days. If your lab is able to support this sort of testing, then this type of testing may be an option. As of September 2021, thirty-four FDA EUAs for antigen tests have been issues; 28 of those 34 include allowances for CLIA-waived testing, and 10 were authorized for home use. Review the FDA list to further examine your options.

The CDC emphasizes that molecular testing remains the "gold standard" for detecting SARS-CoV-2 in a sample, and it "may be necessary to confirm an antigen test result with a laboratory-based NAAT, especially if the result of the antigen test is inconsistent with the clinical context." However, some molecular tests designed for point-of-care testing may not be sufficiently designed for confirmatory testing; consult the instructions for use for any confirmatory test. The CDC makes available two antigen testing algorithms for determining when confirmatory testing is actually recommended.

3.1.4 LAMP and CRISPR

Early on in the pandemic, while PCR was getting most of the attention, reverse transcription loop-mediated isothermal amplification (RT-LAMP), an isothermal nucleic acid amplification technique that allows for RNA amplification, was also quietly being discussed, and it has since gained more attention. In July 2020, the University of Oxford was in the process of getting a rapid, affordable, clinically-validated RT-LAMP test approved for the European market. Oxford also notes that "[a]n advantage of using LAMP technology is that it uses different reagents to most laboratory-based PCR tests." Thi et al. have tested a two-color RT-LAMP assay with an N gene primer set and diagnostic validation using LAMP-sequencing, concluding that the pairing of the two "could offer scalable testing that would be difficult to achieve with conventional qRT-PCR based tests." And California-based Color Genomics set up their own proprietary RT-LAMP system in the summer of 2020, capable of handling up to 10,000 tests per day.

In most cases, LAMP-based testing is much simpler than PCR, lacking the requirement of specialized instruments. Despite LAMP generally being thought of as less sensitive than PCR, the explosion of research into RT-LAMP methods for testing for the presence of SARS-CoV-2 continues to indicate that "under optimized conditions," RT-LAMP methods may actually be able to rival the sensitivity and specificity of many RT-PCR COVID-19 tests. Esbin et al. add:

These methods allow for faster amplification, less specialized equipment, and easy readout. LAMP methods also benefit from the ability to multiplex targets in a single reaction and can be combined with other isothermal methods, like [recombinase polymerase amplification] in the RAMP technique, to increase test accuracy even more. These techniques may be particularly useful for rapid, point-of-care diagnoses or for remote clinical testing without the need for laboratory equipment.

CRISPR methods are also being used in conjunction with RT-LAMP. RT-LAMP creates complementary double-stranded DNA (cDNA) from specimen RNA and then copies (amplifies) it. Then CRISPR methods are used to detect a predefined coronavirus sequence (from a cleaved molecular marker) in the resulting amplified specimen. Though as of September 2021 approved assays using CRISPR-based detection of SARS-CoV-2 are limited to a handful of companies, the technology has some promise as an alternative testing method. CRISPR has the additional advantage of being readily coupled with lateral flow assay technology to be deployed in the point-of-care (POC) setting, though it's worth noting the currently EUAed RT-LAMP-based CRISPR kits are only approved for high-complexity CLIA labs. (The current molecular diagnostic test kits running CRISPR technology are Sherlock BioSciences' Sherlock CRISPR SARS-CoV-2 Kit and Mammoth Biosciences' SARS-CoV-2 DETECTR Reagent Kit, both high-complexity.)

3.1.5 Point-of-care and other alternative testing

Example of a microfluidic chip used in point-of-care medical devices

On September 28, 2020, the WHO published its blueprint for what they call Target Product Profiles (TPP), which "describe the desirable and minimally acceptable profiles" for four different COVID-19 test categories. Addressing POC testing, the WHO recommends that such assays:

• have a sensitivity (true positive rate) of at least 80 percent, with 90 percent or better being desirable;
• have a specificity (true negative rate) of at least 97 percent, with greater than 99 percent being desirable;
• provide results in less than 40 minutes, with less 20 minutes or less being desirable;
• have "a cost that allows broad use, including in low- and middle-income countries";
• be simple enough that only a half day to, optimally, a few hours of training are required to run the test; and
• operate reliably outside a clean laboratory environment.

Though at the time of the announcement few of the available test systems could likely meet all these requirements, it's clear this and other urgencies have put pressure on manufacturers to expand COVID-19 testing to the point of care setting. Additional incentives were offered by the U.S. National Institutes of Health's Rapid Acceleration of Diagnostics (RADx) funding program, which sought to speed up innovation in COVID-19 testing and promote "truly nontraditional approaches for testing that have a slightly longer horizon." In August 2020, RADx had chosen to fund seven biomedical diagnostic companies making new lab-based and POC tests that could significantly ramp up overall testing in the U.S. into September 2020. Four of those offerings were lab-based (from Ginkgo Bioworks, Helix OpCo, Fluidigm, and Mammoth Biosciences) and three were POC tests (from Mesa Biotech, Quidel, and Talis Biomedical), all using varying technologies and methods such as next-generation sequencing, CRISPR, microfluidic chips, nucleic acid testing, antigen testing, and saliva testing. On October 28, 2020, RADx added an additional 15 biomedical diagnostics projects for funding, for a total of 22. As of September 2021, some of those 22 programs have come to fruition, garnering FDA EUAs, including Mesa Biotech's rapid cartridge-based RT-PCR Accula System, Quidel's rapid Sofia SARS Antigen FIA test, Mammoth Bioscience's SARS-CoV-2 DETECTR Reagent Kit, and Visby Medical's COVID-19 Point of Care Test.

Outside the RADx program, enterprising researchers in other parts of the world are also attempting non-traditional approaches to improving COVID-19 testing options. Examples include:

• a method of DNA nanoswitch detection of virus particles;
• a dual biomarker-based finger-stick test for acute respiratory infections;
• a rapid breath test to detect volatile organic chemicals from the lungs;
• an affordable, hand-held spectral imaging device to detect virus in blood or saliva in seconds;
• an ultrahigh frequency spectroscopic scanning device to see virus particles resonating;
• a method that combines optical devices and magnetic particles to detect virus RNA;
• an RNA extraction protocol that uses magnetic bead-based kits;
• a nanotube-based electrochemical biosensor for detecting biomarkers in a sample in less than a minute;
• the additional use of an artificial intelligence (AI) application to better scrutinize test results; and
• the miniaturization of PCR technology to make it more portable and user-friendly.

Of course, most of these are largely experimental technologies, and realistically getting them into the lab may be far out. But they represent out-of-the-box ideas that have some kind of chance at playing a greater role in the clinical laboratory or in point-of-care settings in the future.

3.1.6 Multiplex testing

As the pandemic has progressed and test manufacturers have become more experienced with SARS-CoV-2 test development, multiplex testing has become an option. The multiplex assay—an immunoassay test able to measure multiple analytes in a single test—is certainly not new in itself. In 1989, R. Ekins developed the ambient analyte theory, which stated that miniaturizing an immunoassay can lead to an improved limit of detection (LOD). That work influenced the future development of microarray multiplex technology principles. By 2013, development of multiplex protein immunoassays was becoming increasingly prominent.

As of September 2021, eighteen "multi-analyte" in vitro molecular diagnostic tests are shown as receiving EUAs by the FDA, four of them even authorized for CLIA waived testing. Common additional targets for analysis among the various kits include influenza A, influenza B, and respiratory syncytial virus (RSV). However, several multiplex test kits cover an even broader array of respiratory-affecting organism types and subtypes such as adenovirus and a few other coronavirus types, to name a few. Kits include the ePlex Respiratory Pathogen Panel 2, the NxTAG Respiratory Pathogen Panel + SARS-CoV-2, the QIAstat-Dx Respiratory SARS-CoV-2 Panel, and the BioFire Respiratory Panel 2.1-EZ. (Of the four, the BioFire panel is approved for CLIA waived testing.) Adding multiplex testing of SARS-CoV-2 plus other organisms to your laboratory will largely revolve around your lab's CLIA status and assessment of the available options.

Multiplexing provides a variety of benefits for laboratories and patients. In their 2015 paper on ELISA and multiplex technologies, Tighe et al find that multiplexed immunoassays have the potential to decrease diagnosis times and reduce assay costs. "At the same time, such multiplexing offers more comprehensive analysis whether for research purposes, differential diagnoses, or monitoring of therapeutic interventions." They also note the potential for improved health surveillance of patients, catching early-onset diseases by looking for informative biomarkers. From the perspective of diagnosing infections of SARS-CoV-2 or influenza, the CDC adds that multiplexing helps preserve testing supplies that may be in short supply, conduct more tests in a given time period, and paint a clearer picture of both viruses and their prevalence in a given population.

3.1.7 Variant testing

As the pandemic has progressed, you may have heard talk of a "delta" variant of SARS-CoV-2, which is reportedly more contagious and virulent than the initial strain that kicked off the pandemic. One or more variants of a virus are expected as time progresses, and some of those variants can cause significantly more problems than the source virus. As such, analytical testing of the virus over time is vital to public health.

The purpose of variant testing can be described in two ways, one for public health reasons and another for clinical care reasons. On the public health side, analysis of SARS-CoV-2 variants provides an unbiased, population-level view "of the specific viral strains in circulation and monitors changes in the viral genome over time." With enough public health laboratories conducting this type of analysis—typically whole-genome sequencing (WGS) using next-generation sequencing (NGS) techniques—a clearer picture of how an outbreak spreads is gained, as well as what variants are taking hold and further threatening human populations (even those that are vaccinated). This information is typically shared through the public health system for surveillance and reporting purposes, though the affected patients themselves may never see the data.

On the clinical care side, analysis of SARS-CoV-2 variants provides further insights into improving COVID-19 patient outcomes. Buchan et al. identify three potential insights that clinicians may gain, noting that variant testing allows the clinician:

• to distinguish between an existing, persistent infection caused by one viral strain vs. re-infection by a different viral strain;
• to determine whether a patient not responding to a treatment is affected by a specific viral spike protein (S) gene mutation that is "potentially resistant or less susceptible to neutralizing antibodies or monoclonal antibodies"; and
• to detect in the serum or plasma of a patient post-vaccination "viral S gene substitutions in specific variants that are potentially resistant or less susceptible" to the antibodies the vaccine generates.

If, for example, a patient is diagnosed with a variant that is tied to heightened disease severity, the clinician can opt for additional treatments early on to counteract the variant's effects on the patient. This testing is done in a hospital or reference lab by WGS or by targeting a portion of the genome (e.g., a spike protein) or a specific mutation (using RT-PCR). However, according to Buchan et al., the contributions a mutation makes to a "variant's attributes is not entirely understood, and there is no definitive evidence that directly links a given mutation to poor outcomes, significantly reduced efficacy of SARS-CoV-2 therapies, or vaccine coverage."

That said, and leaving the public health element to the side, if you are a laboratory conducting clinical analyses of SARS-CoV-2 specimens, the likelihood of including viral sequencing and sequence analysis for variant testing may be low for your facility. Such testing is a multi-step process requiring a non-trivial set of resources, often available to large commercial diagnostic laboratories. The CDC represents one possible workflow for genomic sequencing as such:

1. A specimen containing the SARS-CoV-2 virus is received by the lab and promptly entered into the laboratory information system (LIS).
2. The RNA of the SARS-CoV-2 virus is extracted from the sample and then converted to complimentary DNA. It is then enriched and loaded into the appropriate NGS instrument.
3. The instrument runs the sequencing and raw data is collected, with the lab maintaining quality control steps. The raw data is turned into actionable sequence data.
4. The sequence data is verified for suitability, with a resequencing occurring if found to be inadequate. Otherwise, the data is then analyzed and interpreted.
5. The final approved sequencing results are reported to the appropriate state, local, tribal, or territorial public health department.

If your diagnostic lab has or is planning on adding sequencing tools to supplement clinical diagnostics, it may make sense to consider adding variant testing to your available options. But in reality, this sort of testing may largely be left to large institutions, such as the University of Rochester Vaccine Treatment Evaluation Unit or the Yale School of Public Health.

3.2 What kind of space, equipment, and supplies will you need?

3.2.1 Laboratory space arrangements

PCR considerations

Whether adding PCR to your existing laboratory, modifying existing PCR workflows, or starting from scratch, preventing contamination is a top priority. As PCR can effectively amplify even the tiniest of quantities of DNA and RNA, the risk of amplifying a contaminant and ruining the validity of an assay is very real. Contamination typically comes from non-amplified environmental substances such as aerosols, and from carryover contamination of amplicons from earlier PCR cycles. As such, not only do best-practice processes and procedures (P&P) need to be followed (e.g., unidirectional workflow, thorough cleaning procedures, proper preparation and disposal), but also where to place PCR-related equipment must be carefully considered.

When possible, separate rooms for sample preparation, PCR setup, and post-PCR activities, each with their own airflow control, are encouraged. However, the laboratory attempting to add PCR to an already small clinical diagnostic lab may not have the luxury of having multiple rooms. In that case, a single-room setup may suffice, if the workflow areas remain demarcated or physically partitioned. Additionally, a single-room setup must also have stricter P&P and design controls to offset the space constraints. For example, the sample preparation area of the room should have a laminar flow hood with UV light that is regularly cleaned, and post-PCR analysis may need to occur later in the day after cleanup from prior steps. Of course, always maintaining unidirectional workflow—regardless of number of rooms—is also critical to minimizing contamination. For example, technicians shouldn't be transporting amplified materials into the DNA extraction area.

Although dated, Roche Diagnostics' 2006 PCR Applications Manual provides a detailed breakdown of setting up the laboratory for PCR. Das et al. and Dr. Jennifer Redig provide additional valuable insight. The World Health Organization (WHO) also provides guidance for setting up molecular testing in the lab.

Isothermal amplification considerations

Similarly, because DNA and RNR amplification is involved, contamination concerns exist with isothermal amplification techniques. Multiple pipetting steps and repeated freezing and thawing of reagents can still lead to cross-contamination, as does opening the reaction chamber after reaction is completed. However, the advent of microfluidics and lateral flow technologies in isothermal amplification processes has seen the development of "fully enclosed microstructured devices into which performing the isothermal amplification reduces the risk of sample contamination and allows integration and portable device realization." Even more cutting-edge techniques to reduce contamination such as the CUT-LAMP technique of Bao et al. or the dUTP/UDG system for COVID-19 RT-LAMP reactions of Kellner et al. hold further promise in making isothermal amplification processes in the laboratory easier to manage. That said, labs running isothermal amplification processes such as LAMP requiring analysis with agarose gel electrophoresis or a method requiring the opening of reaction vessels will preferably have a secondary area set up for analysis steps so as to minimize the chances of contamination.

3.2.2 Instruments and assays

Eppendorf Mastercycler Pro S, a thermal cycler for PCR and other applications

High- and moderate-complexity CLIA testing

Thermal cyclers are the standard instruments for PCR testing. Today, real-time or quantitative (qPCR) systems largely fill this niche. However, digital and droplet digital PCR systems are emerging, and they have the benefit of producing even more rapid, precise, sensitive, accurate, and reproducible results, and they are capable of direct quantification and multiplexing. Other instruments and accessories for PCR workflows include proper power supplies, analytical balances, electrophoresis chambers, water and/or dry baths, and mini/micro centrifuges. However, if you're considering the addition of PCR workflow to your laboratory, the thermal cycler is typically where the largest up-front cost will be. As such, it's important to ask yourself critical questions to help guide your acquisition decisions.

As part of their June 2018 survey on PCR equipment, Lab Manager posed five questions potential buyers should ask before making PCR purchases:

1. Do your current and long-term needs require basic PCR systems, qPCR systems, or digital PCR systems?
2. What sample formats do you anticipate using?
3. What throughput requirements do you have now and anticipate in the near future?
4. What are you willing to sacrifice in regards to temperature ramp up and cool down times and accuracies?
5. Do you anticipate needing to run more than one independent PCR at the same time (multiblock PCR)?

Given the considerable investment that goes into these and other life science instruments, you may want to seek vendors who have a strong track record of supporting and supplying parts for instruments they manufacture and distribute years after the instruments are introduced.

As for PCR-based assays, the U.S. FDA has issued EUAs for more than 200 of them. The most up-to-date listing is of course found at the FDA website. However, sorting through the extra details can be tedious. The Center for Systems Biology at Harvard has been maintaining a contextual PDF chart of the various COVID-19 diagnostic tests, which includes information such as run time, manufacturer-supplied data, and published clinical data (when available). This may prove useful in deciding on one or more particular tests. As with many aspects of this pandemic, other factors that may influence your choice of test kit include overall availability, cost, reagents included with the assay, and reagents separately required and their availability.

Isothermal amplification techniques have the advantage of not requiring an expensive thermal cycler. Instrument-appropriate reaction vessels, baths, heating units, turbidimeters, thermocyclers, etc. may be required, depending on what type of amplification you're doing. Companies like Meridian Bioscience offer LAMP-based molecular platforms, though they may not offer a specific COVID-19 assay to run on the platform. As can be seen in Table 1, two isothermal amplification assays that run on their own proprietary instrument have received EUAs and are CLIA-waved, with a third potentially on the way. Using these systems and their COVID-19 assays at the point of care provides a somewhat more attractive option for laboratories wanting to add COVID-19 or even multiplex viral assays to their offerings.

CLIA-waived testing

If you're running a POL, or attempting to provide COVID-19 testing at the point of care, you'll be looking at the following molecular diagnostic assays (Table 1) and antigen diagnostic assays (Table 2), depending on the level of accuracy and purpose of use required in your POL. Remember that—broadly speaking—antigen tests tend to have lower sensitivities than molecular tests, leaving antigen tests best used as surveillance or repeat screening tools. For example, the directions for the BD Veritor System for Rapid Detection of SARS-CoV-2 manufactured by Becton, Dickinson and Company state: "Sensitivity of the test after the first five days of the onset of symptoms has been demonstrated to decrease as compared to a RT-PCR SARS-CoV-2 assay."

Table 1. CLIA-waived COVID-19-related in vitro molecular diagnostic tests (e.g., RT-PCR, LAMP, isothermal amplification) receiving U.S. FDA Emergency Use Authorizations (EUAs) First date EUA issued Manufacturer Name of test or assay Required instrument Technology (Method) Multi-analyte? RADx-funded? Approved for at-home? Additional comments 20 March 2020 Cepheid Xpert Xpress SARS-CoV-2 test GeneXpert Xpress System (Tablet and Hub Configurations) Molecular (RT-PCR) No No No Has largely received positive review of sensitivity and specificity. 23 March 2020 Mesa Biotech Inc. Accula SARS-CoV-2 test Accula Dock or the Sekisui Diagnostics Silaris Dock (discontinued) Molecular (RT-PCR) No Yes No Has received only minor scrutiny, with only several dozen FDA complaints/reports 27 March 2020 Abbott Diagnostics Scarborough, Inc. ID NOW COVID-19 ID NOW Molecular (isothermal amplification) No No No Targets "a unique region of the RNA-dependent RNA polymerase (RdRP) gene."Device and test were target of FDA scrutiny due to sensitivity issues reported in 2020 and into 2021 In October 2020, Abbott released additional study data showing overall sensitivity of 93.3% and specificity of 98.4%, emphasizing the ID NOW's best use with samples taken within seven days of symptom onset. In 2020, some 393 complaints were reported to the FDA, with 1,492 complains being reported in 2021 (through July 31) according to an FDA MAUDE (Manufacturer and User Facility Device Experience) search. On August 27, 2021, the FDA re-issued its EUA for the ID NOW with updated in silico inclusivity analysis results (among other things), but it's not clear if the FDA is continuing to work with Abbott on the test's accuracy claims. 10 June 2020 Cue Health Inc. Cue COVID-19 Test Cartridge Cue Health Monitoring System Molecular (isothermal amplification) No No No "Test primers amplify the nucleocapsid (N) region of the gene" 14 September 2020 Roche Molecular Systems, Inc. cobas SARS-CoV-2 & Influenza A/B Assay cobas Liat PCR System Molecular (RT-PCR) Yes No No Everitt et al. offer some discussion and citations concerning research related to the cobas LIAT PCR system and its assays. 24 September 2020 Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV test GeneXpert Xpress System (Tablet and Hub Configurations) Molecular (RT-PCR) Yes No No Development of this multiplex assay for SARS-CoV-2, Flu A, Flu B, and RSV was announced in June 2020. After receiving its EUA in September 2020, received advanced development support through the Department of Health and Human Services and the Department of Defense. 02 October 2020 BioFire Diagnostics, LLC BioFire Respiratory 2.1 (RP2.1) Panel BioFire FilmArray Systems Molecular (RT-PCR) Yes No No From the manufacturer: "The BioFire RP2.1 Panel (EUA) detects 22 respiratory pathogens, including SARS-CoV-2, to help clinicians quickly rule in and rule out common causes of respiratory illness in about 45 minutes." Creager et al. reported their evaluation findings in the Journal of Clinical Virology, stating that the panel "has similar performance to high throughput assays used for the detection of COVID-19." 17 November 2020 Lucira Health, Inc. Lucira COVID-19 All-In-One Test Kit N/A Molecular (RT-LAMP) No No Yes First complete at-home COVID test kit receiving EUA. For CLIA-waived labs and prescription at-home use. Test device is apparently one-time-use and not reusable. 27 November 2020 Cepheid Xpert Omni SARS-CoV-2 test GeneXpert Omni System Molecular (RT-PCR) No No No For CLIA-waived testing, the test is limited to nasopharyngeal, anterior nasal, or mid-turbinate swab specimens. Product status unclear, as it was listed on website in January 2021, but not listed there as of September 2021. 08 February 2021 Visby Medical, Inc. Visby Medical COVID-19 Point of Care Test N/A Molecular (RT-PCR) No Yes No "By shrinking rapid PCR technology to palm-sized dimensions and eliminating the need for an additional instrument or reader, Visby Medical’s test provides fast, accurate, and actionable results at the point of need." 05 March 2021 Cue Health Inc. Cue COVID-19 Test for Home and Over The Counter Use Cue Health Monitoring System Molecular (isothermal amplification) No No Yes Described as "the nation’s first molecular diagnostic test available without a prescription to consumers for home use and to enterprise users and healthcare professionals without CLIA certification." It is also able to be used for screening purposes. 09 April 2021 Lucira Health, Inc. Lucira CHECK-IT COVID-19 Test Kit N/A Molecular (RT-LAMP) No No Yes Appears to be an over-the-counter (vs. prescription) version of its Lucira COVID-19 All-In-One Test Kit from November 2020. Also able to be used for screening. 17 June 2021 Roche Molecular Systems, Inc. cobas SARS-CoV-2 Assay cobas Liat PCR System Molecular (RT-PCR) No No No Appears to be similar to its multi-analyte product from 2020 but solely for COVID-19, and also able to be used for screening. N/A (Anticipated) Talis Biomedical Talis One Cartridge Talis One Instrument Molecular (RT-LAMP) No Yes To be determined Expectations are that it will receive an FDA EUA and be CLIA-waived, but yet to be determined. As of August 2021, it was still awaiting FDA authorization.

Table 2. CLIA-waived COVID-19-related in vitro antigen diagnostic tests (e.g., lateral flow, immunoassay, etc.) receiving U.S. FDA Emergency Use Authorizations (EUAs); OTC = Over the counter First date EUA issued Manufacturer Name of test or assay Required instrument Technology (Method) Multi-analyte? RADx-funded? Approved for at-home? Additional comments 08 May 2020 Quidel Corporation Sofia SARS Antigen FIA Sofia 2 Antigen (Lateral flow) No Yes No SARS-CoV-2 Sensitivity (PPA): 96.7%; SARS-CoV-2 Specificity (NPA): 100% 02 July 2020 Becton, Dickinson and Company BD Veritor System for Rapid Detection of SARS-CoV-2 BD Veritor Plus Antigen (Immunoassay) No No No SARS-CoV-2 Sensitivity (PPA): 83.9%; SARS-CoV-2 Specificity (NPA): 100% 18 August 2020 LumiraDx UK Ltd. LumiraDx SARS-CoV-2 Ag Test LumiraDx Platform Antigen (Microfluidic) No No No SARS-CoV-2 Sensitivity (PPA): 97.6%; SARS-CoV-2 Specificity (NPA): 96.6%. As of September 2021, at least one performance evaluation study is in-process. 26 August 2020 Abbott Diagnostics Scarborough, Inc. BinaxNOW COVID-19 Ag Card NAVICA mobile system Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 84.6%; SARS-CoV-2 Specificity (NPA): 98.5% 02 October 2020 Quidel Corporation Sofia 2 Flu + SARS Antigen FIA Sofia 2 Antigen (Lateral flow) Yes Yes No SARS-CoV-2 Sensitivity (PPA): 95.2%; SARS-CoV-2 Specificity (NPA): 100% 08 October 2020 Access Bio, Inc. CareStart COVID-19 Antigen N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 90.47%; SARS-CoV-2 Specificity (NPA): 99.66% (Note: average of swab types) 07 December 2020 Luminostics, Inc. Clip COVID Rapid Antigen Test Clip Analyzer (PDF) Antigen (Lateral flow) No Yes No SARS-CoV-2 Sensitivity (PPA): 96.9%; SARS-CoV-2 Specificity (NPA): 100% 15 December 2020 Ellume Limited Ellume COVID-19 Home Test A mobile phone that supports their app Antigen (Lateral flow) No Yes Yes (OTC) SARS-CoV-2 Sensitivity (PPA): 94.6%; SARS-CoV-2 Specificity (NPA): 96.9% 16 December 2020 Abbott Diagnostics Scarborough, Inc. BinaxNOW COVID-19 Ag Card Home Test A mobile phone that supports their NAVICA app Antigen (Lateral flow) No No Yes (Prescription) SARS-CoV-2 Sensitivity (PPA): 91.7%; SARS-CoV-2 Specificity (NPA): 100% 18 December 2020 Quidel Corporation QuickVue SARS Antigen Test N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 96.6%; SARS-CoV-2 Specificity (NPA): 99.3% 04 February 2021 Princeton BioMeditech Corp. Status COVID-19/Flu A&B N/A Antigen (Lateral flow) Yes No No SARS-CoV-2 Sensitivity (PPA): 93.9%; SARS-CoV-2 Specificity (NPA): 100% 01 March 2021 Quidel Corporation QuickVue At-Home COVID-19 Test N/A Antigen (Lateral flow) No No Yes (Prescription) SARS-CoV-2 Sensitivity (PPA): 84.8%; SARS-CoV-2 Specificity (NPA): 99.1% 31 March 2021 Quidel Corporation QuickVue At-Home OTC COVID-19 Test N/A Antigen (Lateral flow) No No Yes (OTC) SARS-CoV-2 Sensitivity (PPA): 83.5%; SARS-CoV-2 Specificity (NPA): 99.2% 31 March 2021 Abbott Diagnostics Scarborough, Inc. BinaxNOW COVID-19 Ag Card 2 Home Test N/A Antigen (Lateral flow) No No Yes SARS-CoV-2 Sensitivity (PPA): 91.7%; SARS-CoV-2 Specificity (NPA): 100% 16 April 2021 Celltrion USA, Inc. Celltrion DiaTrust COVID-19 Ag Rapid Test N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 93.33%; SARS-CoV-2 Specificity (NPA): 99.03% 06 May 2021 InBios International, Inc. SCoV-2 Ag Detect Rapid Test N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 86.67%; SARS-CoV-2 Specificity (NPA): 100% 20 May 2021 Salofa Oy Sienna-Clarity COVID-19 Antigen Rapid Test Cassette N/A Antigen (Lateral flow) No No No SARS-CoV-2 Relative Sensitivity: 87.5%; SARS-CoV-2 Relative Specificity: 98.9% (note that it's relative) 04 June 2021 OraSure Technologies, Inc. InteliSwab COVID-19 Rapid Test N/A Antigen (Lateral flow) No No Yes (OTC) SARS-CoV-2 Sensitivity (PPA): 84.3%; SARS-CoV-2 Specificity (NPA): 97.9% 04 June 2021 OraSure Technologies, Inc. InteliSwab COVID-19 Rapid Test Rx N/A Antigen (Lateral flow) No No Yes (Prescription) SARS-CoV-2 Sensitivity (PPA): 84.3%; SARS-CoV-2 Specificity (NPA): 97.9% 04 June 2021 OraSure Technologies, Inc. InteliSwab COVID-19 Rapid Test Pro N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 84.3%; SARS-CoV-2 Specificity (NPA): 97.9% 08 July 2021 Ellume Limited ellume.lab COVID Antigen Test ellume.lab digital device Antigen (Lateral flow) No Yes No SARS-CoV-2 Sensitivity (PPA): 81.8%; SARS-CoV-2 Specificity (NPA): 100% 13 July 2021 GenBody Inc. GenBody COVID-19 Ag N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 96.0%; SARS-CoV-2 Specificity (NPA): 99.28% 28 July 2021 PHASE Scientific International, Ltd. INDICAID COVID-19 Rapid Antigen Test N/A Antigen (Lateral flow) No No No SARS-CoV-2 Sensitivity (PPA): 84.4%; SARS-CoV-2 Specificity (NPA): 96.6% (average of health-care-provider-collected and self-collected) 02 August 2021 Access Bio, Inc. CareStart COVID-19 Antigen Home Test (Not on website yet) N/A Antigen (Lateral flow) No No Yes (OTC) SARS-CoV-2 Sensitivity (PPA): 86.6%; SARS-CoV-2 Specificity (NPA): 97.6% (Note: average of swab types) 24 August 2021 Becton, Dickinson and Company BD Veritor At-Home COVID-19 Test (Not on website yet) N/A Antigen (Lateral flow) No No Yes SARS-CoV-2 Sensitivity (PPA): 84.6%; SARS-CoV-2 Specificity (NPA): 99.8%

3.2.3 Reagents

High- and moderate-complexity CLIA testing

At various times during the pandemic, reagent shortages have hampered many efforts to expand testing in parts of the world, including the United States. For the attentive laboratory wanting to remain agile in its testing, the laboratory's reagent choices will likely be closely tied to both the assays it chooses to implement and how reliably the supplier can get them to the lab. This in turn is likely driven by whether the lab is using a lab-developed test or a test kit. In some cases, e.g., the Xiamen Zeesan Biotech SARS-CoV-2 Test Kit (Real-time PCR), all but the Virus RNA Extraction Kit is included. On the other hand, Biomeme's SARS-CoV-2 Real-Time RT-PCR Test requires the separate acquisition of PCR buffer and external controls other than the exogenous RNA Process Control that comes with the kit. Yale's SalivaDirect is a more flexible test, validated for use with multiple instruments and reagents that are not proprietary to Yale. Pay close attention to what comes with the assay, typically by reviewing the instructions for use (IFU; found on the FDA's EUA page).

For PCR, the five basic reagents are template DNA, PCR primers, nucleotides, PCR buffer, and thermostable DNA polymerase. Some of these components can be acquired pre-mixed as a "master mix." For example, Thermo Fisher's PCR Master Mix contains a thermostable DNA polymerase called Taq, nucleotides called deoxynucleotide triphosphates (dNTPs), and a buffer, which "saves time and reduces contamination due to a reduced number of pipetting steps."

Reagent cost and usage for isothermal amplification methods such as LAMP are similar, though buffers and primers specific to the method are required.

CLIA-waived testing

The FDA EUA devices (Table 1 and 2) all come with the necessary reagents, with the exception of any controls or references you may require. Refer to the IFU for the waived test kit to determine what additional consumables you'll require.

3.2.4 Consumables

High- and moderate-complexity CLIA testing

Non-reagent consumables for high- and moderate-complexity CLIA testing include PCR tubes and plates; pipettes and tips; films, foils, and sealing mats; swabs; and viral transport media, among others. Some like Kellner et al. have experimented with methods to make isothermal amplifications methods more approachable in resource-poor environments by, for example, developing a pipette-free version of LAMP.

CLIA-waived testing

The FDA EUA devices (Table 1 and 2) may require a few extra consumables. For example, the Accula SARS-CoV-2 test kit comes with swabs and the Xpert Xpress SARS-CoV-2 kit comes with disposable transfer pipettes. Refer to the IFU for the waived test kit to determine what additional consumables you'll require.

3.2.5 Software and services

A June 2020 report by Weemaes et al. in the Journal of the American Medical Informatics Association describes the bottlenecks they encountered in their test workflows at the Belgian National Reference Center, and how they updated their LIS with functionality to resolve those bottlenecks. In addition to adding a COVID-19–specific order set into the computerized physician order entry (CPOE) module integrated with both their LIS and electronic health record (EHR), they included an up-to-date triage criteria component, a tool for optimizing sampling and packaging, a COVID-19 status button, and improved reporting modules for automating reference testing and epidemiological reporting. They also added extra database and data mining functionality to facilitate research and insights into epidemiologies and treatments. Their conclusion: "Rapidly developed, agile extendable LIS functionality and its meaningful use alleviates the administrative burden on laboratory personnel and improves turnaround time of SARS-CoV-2 testing." The Association of Public Health Laboratories comes to a similar conclusion in regard to laboratory informatics solutions and public health laboratories' COVID-19 testing.

As such, adding COVID-19 and other respiratory illness testing to your workflow may necessitate an information management system, or an upgrade of your existing software systems. You may experience many of the same bottlenecks the Belgian National Reference Center experienced, especially if you're still working primarily with paper-based test ordering. Those researchers found that paper-based COVID-19 test requests often:

• omitted critical clinical status and contact information;
• slowed down epidemiological and research studies;
• hindered proper pre-analytical biosafety procedures; and
• impeded rapid response to evolving test criteria and clinical insights through test ordering protocols.

How interoperable your laboratory software solution is with other systems such as EHRs is also worth consideration. The next chapter addresses system interoperability in greater detail, but it's worth mentioning it here in the context of adding software to improve testing workflows for SARS-CoV-2 and other respiratory viruses. Broadly speaking, improving interoperability among clinical informatics systems—whether at the point of care or within a specific laboratory—is recognized as an important step towards improving health outcomes. However, while developers of EHRs and other clinical informatics systems have intended to improve their software's interoperability, the COVID-19 pandemic has, at times, unfortunately shown the inadequacies still inherent in that software's overall design. As such, any research into acquiring a laboratory information management system (LIMS), LIS, or other clinical information management solution should take into account how well that solution is able to integrate with your other clinical systems, as well as any other third-party systems like physician or hospital EHRs. And it's not just the software solutions you'll want to consider. Will the new instruments you add for getting your lab rolling with clinical respiratory illness testing integrate with your software?

Finally, although rare, you may find you don't have the in-house expertise to fully implement a COVID-19 testing line to your laboratory. In such a case, you may need to turn to a laboratory services consultancy with experience in SARS-CoV-2 test method validation, instrument procurement and implementation, and legal matters. (See the next section for a representative example of consultants advertising COVID-19 testing knowledge and services for labs.)

3.2.6 Major vendors and consultants

Table 3 lists the major vendors developing and selling PCR, isothermal amplification, and NGS supplies, instruments, and software for both clinical diagnostics and life science research. "Research use only" equipment like Siemens Healthcare's Fast Track Cycler was ignored for completing the table. The vendor list was largely compiled from vendors identified in a handful of online market reports on PCR, with an added sprinkling of a few additional reagent vendors (e.g., Jena Bioscience, LGC, and New England BioLabs) who address isothermal amplification supplies in addition to PCR. Note that this is not an endorsement for any particular vendor.

Table 3. Major players operating in the global diagnostic PCR, isothermal amplification, and NGS market Vendor Shop all products PCR machines Nucleic acid extraction and purification machines Immunoassay analyzers and assays PCR and qPCR assays PCR and qPCR enzymes and master mixes DNA/RNA purification, quantitation, and amplification supplies LAMP assays Isothermal amplification enzymes and master mixes NGS supplies Supporting labware and supplies Software Abbott Abbott products ✓ - - ✓ ✓ - - - - ✓ ✓ Agilent Technologies Agilent products ✓ - - ✓ ✓ ✓ - - ✓ ✓ ✓ Becton, Dickinson and Company BD products ✓ - - ✓ - - - - - ✓ - Bio-Rad Laboratories, Inc. Bio-Rad products ✓ - ✓ ✓ ✓ ✓ - - ✓ ✓ - bioMérieux SA bioMérieux products - ✓ ✓ ✓ - - - - - ✓ ✓ Danaher Corporation and its companies Cepheid products, Beckman Coulter products, and Beckman Life Sciences products ✓ - ✓ ✓ - ✓ - - - ✓ - F. Hoffmann-La Roche AG Roche Diagnostics products ✓ ✓ - ✓ ✓ ✓ - - ✓ - ✓ Jena Bioscience GmbH Jena Bioscience products - - - ✓ ✓ ✓ ✓ ✓ ✓ - - LGC Limited and its companies Lucigen products - - - ✓ ✓ ✓ - ✓ ✓ - - Merck KGaA Sigma-Aldrich products and Millipore Sigma products - - - ✓ ✓ ✓ - - ✓ ✓ - New England BioLabs, Inc. NEB products - - - ✓ ✓ ✓ ✓ ✓ ✓ - - Promega Promega products and catalog - ✓ - ✓ ✓ ✓ - - ✓ - - QIAGEN N.V. QIAGEN products ✓ ✓ - ✓ ✓ ✓ - - ✓ - ✓ Siemens Healthcare GmbH Siemens Healthcare products - ✓ - ✓ - ✓ - - - - ✓ Thermo Fisher Scientific, Inc. Thermo Fisher products and Fisher Scientific products ✓ ✓ - ✓ ✓ ✓ - ✓ ✓ ✓ ✓

The "Software" column of Table 3 represents whether or not the vendor offers laboratory informatics software such as a LIMS or LIS. Those vendors' solutions may or may not be tailored to handle the specific requirements of a clinical diagnostic or virology lab handling COVID-19 and other viruses. (See the next chapter for more in-depth information about working an informatics solution into COVID-19 and other viral testing workflow.) A non-endorsed, representative example of vendors who do include:

• Abbott Informatics Corporation - STARLIMS LIMS
• AgileBio - COVID-19 LC
• Autoscribe Informatics, Inc. - Matrix Covid LIMS
• BioSoft Integrators, LLC - LabOptimize LIMS for COVID-19
• CloudLIMS.com, LLC - CloudLIMS for COVID-19
• Common Cents Systems, Inc. - Apollo LIMS for COVID-19
• Comp Pro Med, Inc. - COVID-19 Rapid Deployment LIS
• Illumina, Inc. - BaseSpace Clarity LIMS for COVID-19
• Junction Concepts, Inc. - QBench
• LabLynx, Inc. - COVIDLiMS
• LabVantage Solutions, Inc. - LabVantage COVID-19 LIMS
• Physion, LLC - Ovation LIMS for COVID-19
• Vendor:Sunquest Information Systems, Inc. - Mitogen LIMS for COVID-19 (PDF)
• Third Wave Analytics, Inc. - Lockbox LIMS for SARS-CoV-2 Clinical Testing

Additionally, when in-house knowledge is lacking, a consultant may be required. These consultants are meant to be representative examples of those laboratory consulting firms indicating they have the knowledge to help a laboratory with COVID-19-related testing and other issues. This list is not an endorsement for any particular consultant:

• ARUP Laboratories (Utah)
• Colaborate (Florida)
• Elite Diagnostics (North Carolina)
• Freed Associates (California)
• Lighthouse Lab Services (North Carolina)
• Optimum Healthcare IT (Florida)
• The COVID Consultants (Colorado)
• Triumverate Environmental (Massachusetts)

3.3 What other considerations should be made?

3.3.1 U.S. regulatory compliance

In the previous chapter, the regulatory hurdles of the Health Insurance Portability and Accountability Act (HIPAA) and the Clinical Laboratory Improvement Amendments (CLIA) were addressed. For those laboratories that are already operating in the clinical laboratory sphere, it should be relatively simple to address the additional considerations and pandemic-specific changes to those two regulations as described previously. In addition to that information, you can always periodically check the U.S. Department of Health & Human Services' (HHS') Office for Civil Rights and their COVID-19 announcements and guidance, as well as the Centers for Medicare & Medicaid Services (CMS) emergencies page.

If for some reason you're not a clinical lab—or want to start a new lab—and want to take on COVID-19 and other clinical testing, you're going to need to get fast tracked into the CLIA program, for starters. Fortunately, CMS has already displayed a willingness to help labs wanting to perform COVID-19 testing receive their CLIA certificate rapidly. Form CMS-116 will need to be completed and submitted to your state survey agency contact. Of course, while you're waiting, you'll also want to become familiar with the trappings of CLIA by tapping into resources like the CMS page for CLIA, CDC page for CLIA, and resources available from professional organizations like the American Academy of Family Physicians. If all goes as planned, and directions are followed, you should have your CLIA certificate in no time. CMS adds:

We want to ensure that laboratories located in the United States applying for a CLIA certificate are able to begin testing for COVID-19 as quickly as possible. Once the laboratory has identified a qualified laboratory director and has provided all required information on the CMS-116 application, a CLIA number will be assigned. Once the CLIA number has been assigned, the laboratory can begin testing as long as applicable CLIA requirements have been met (e.g., establishing performance specifications).

On the HIPAA side of things, you'll want to tap into resources such as the HHS' HIPAA training materials and resources, as well as their previously mentioned COVID-19 announcements and guidance.

Other considerations include:

• taking the time to get accredited to ISO 15189:2012 Medical laboratories — Requirements for quality and competence, "used by medical laboratories in developing their quality management systems and assessing their own competence";
• understanding and training on packaging (e.g., UN3373 Biological Substance, Category B) and shipping COVID-19 specimens (e.g., International Air Transport Association (IATA) Dangerous Goods Regulations), if you will be conducting such activities;
• understanding the significance of and validating workflow procedures to at least Biosafety Level 2 (note there is no single U.S. government entity which has total responsibility for enforcing biosafety levels); and
• understanding and training on Occupational Safety and Health Administration (OSHA) requirements for laboratory workers and employers for COVID-19.

3.3.2 Reporting

The topic of reporting COVID-19 results to local and regional health departments—as well as any internal medical reporting—is covered in detail in the next chapter. You'll want to be sure you and your team understand your state's health department reporting requirements, as well as what code sets (e.g., LOINC, SNOMED, ICD, and CPT) to use. The success of epidemiologists' response to outbreaks and pandemics depends on quality data reporting, and using the correct code sets helps labs meet reporting requirements, as well as ensure proper payment. The U.S. Centers for Disease Control and Prevention provides guidance on how to report COVID-19 laboratory data, which also makes for necessary reading.

3.3.3 Billing, Medicare, and Medicaid

The COVID-19 pandemic has unquestionably put the U.S. health care system in a tough spot. That health care system, with all its warts, has arguably not done well to handle so many unanticipated health issues from a broad portion of the population. From a provider side, proper reimbursement for COVID-19 testing is among the many issues that must be addressed. One key aspect of ensuring proper reimbursement in a reasonable time frame is first making sure a clear preregistration process that captures critical patient and facility information is conducted. (This can be facilitated and made easier as a first-step process in a clinical informatics solution, for example.) Critical patient and facility information includes (but is not limited to):

• name, date of birth, and gender
• race and ethnicity
• demographic information such as full address and phone number
• ordering physician or attending health care provider for test (if applicable)
• facility's National Provider Identifier (NPI)
• patient insurance company name, policy ID, group ID, insured's name, and insured relationship to patient (if insured)
• whether or not it's the patient's first test (federal reporting requirement)
• whether or not the patient is a resident of a congregate care setting (federal reporting requirement; also, e.g., additional Medicaid reimbursement may be available in some states)
• whether or not the patient is a healthcare worker (federal reporting requirement; also, e.g., may affect the patient's worker's compensation claim)
• whether or not the patient is pregnant (federal reporting requirement; also, e.g., Medicare will only accept a COVID-19 code as secondary if the primary diagnosis code is viral disease complicating pregnancy, childbirth, or puerperium)

Secondarily, it's also important to have a plan in place for testing the uninsured. While the Families First and Coronavirus Relief Act (FFCRA) and the National Disaster Management System (NDMS) have historically provided legal mechanisms for reimbursement for what should otherwise be free patient testing for SARS-CoV-2 and the associated visit, ambiguities of these mechanisms and how they were enforced still managed to cause problems. For example, while providers could turn to the NDMS (until funds ran out) to pay uninsured claims at 110% of Medicare rates—with states' opting to cover those costs through their Medicaid program—providers were not obligated by the law to seek reimbursement from those entities and could optionally bill the uninsured patient directly, which was against the spirit of the FFCRA. Given these past problems and any lingering questions about existing programs like the HHS and HRSA coverage assistance programs, it's important to know what your lab's policy will be on managing uninsured patient claims. How will you get reimbursed if you're accepting uninsured patients? Resources that may help with these decisions include the Health Resources & Services Administration's information page and associated FAQ.

For Medicare, Medicaid, and otherwise insured patients, the lab will likely have (or presumably acquire) someone on hand with billing experience. However, the preregistration information previously mentioned will still be important to implement. And staying up-to-date regarding billing issues is also important (e.g., CMS' October 2020 announcement about payment for high-throughput COVID-19 tests and turnaround times

For further guidance on billing issues, you may wish to consult with CMS' extensive document titled COVID-19 Frequently Asked Questions (FAQs) on Medicare Fee-for-Service (FFS) Billing. Also, the next chapter addresses code sets for reporting and billing, which may prove useful.

3.3.4 Biosafety

Like any other communicable disease, laboratories handling specimens that are suspected or confirmed of containing the SARS-CoV-2 virus must take appropriate precautions to protect all stakeholders. This involves not only any in-house protocols for preventing contamination but also any official guidance that goes beyond or supersedes in-house protocols. Examples of guidance documents include the World Health Organization's Laboratory biosafety guidance related to coronavirus disease (COVID-19), the CDC's Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19), and the CDC's Guidance for General Laboratory Safety Practices during the COVID-19 Pandemic. Additionally, it may be helpful to look to what other laboratories are doing. In a brief article published in The Lancet Microbe, Choy highlights an International Federation of Clinical Chemistry and Laboratory Medicine Taskforce survey of biochemistry labs and how they've been mitigating biohazard risks associated with SARS-CoV-2. Actions include:

• restricting laboratorian access to testing of suspected and confirmed COVID-19 patient samples;
• tightening of delivery and shipping procedures of suspected and confirmed COVID-19 patient samples;
• limiting add-on test requests for suspected and confirmed COVID-19 patients;
• increasing the frequency of disinfection; and
• considering the expanded use of autoclaving before sample disposition.

Additional aspects of operations that laboratory managers may wish to implement include "number of shifts per day, the number of staff per shift, total number of staff accessible to work in the laboratory, shift change frequency, team-splitting arrangements, and fixed work–rest days." Arranging staff into smaller teams while reducing the consecutive number of shifts worked may reduce risks; however, managers of labs struggling to meet turnaround times may feel like this isn't realistically possible. In the end, the safety of personnel must be of highest importance, even while trying to rapidly and accurately conduct COVID-19 testing.